Cell therapy methods have been developed in order to enhance the host immune response to tumors, viruses and bacterial pathogens. Cell therapy methods often involve the ex-vivo activation and expansion of T-cells. Examples of these type of treatments include the use of tumor infiltrating lymphocyte (TIL) cells (see U.S. Pat. No. 5,126,132 issued to Rosenberg), cytotoxic T-cells (see U.S. Pat. No. 6,255,073 issued to Cai, et al.; and U.S. Pat. No. 5,846,827 issued to Celis, et al.), expanded tumor draining lymph node cells (see U.S. Pat. No. 6,251,385 issued to Terman), and various other lymphocyte preparations (see U.S. Pat. No. 6,194,207 issued to Bell, et al.; U.S. Pat. No. 5,443,983 issued to Ochoa, et al.; U.S. Pat. No. 6,040,177 issued to Riddell, et al.; U.S. Pat. No. 5,766,920 issued to Babbitt, et al.).
T-cells must be activated in order to proliferate, perform effector functions and produce cytokines (Liebowitz, Lee et al. 1998). T-cells require direct contact with antigen presenting cells (“APC”) for activation. APC convert protein antigens to peptides and then present peptide-MHC complexes in a form that can be recognized by T-cells. The interaction of the peptide-MHC complex on the APC and the T-cell receptor (“TCR”) on the surface of the T-cell usually provides the first of the two signals required for activation. The second of the two signals required for activation is usually provided by membrane-bound or secreted products of the APC.
Due to the difficulty in maintaining large numbers of natural APC in cultures and in identifying disease-associated antigens and controlling the processing and presentation of these antigens to T-cells by natural APC, alternative methods have been sought for ex-vivo activation of T-cells for use in cell therapy. One method is to by-pass the need for the peptide-MHC complex on natural APC by instead stimulating the TCR with polyclonal activators, such as immobilized or cross-linked anti-CD3 monoclonal antibodies (mAbs) to provide the first signal to T-cells. Other methods take advantage of the secondary T-cell activation pathway to provide the first signal, such as the use of immobilized or cross-linked anti-CD2 mAb.
The combination of anti-CD3 mAb (first signal) and anti-CD28 mAb (second signal) is most commonly used to substitute for natural APCs in inducing T-cell activation in cell therapy protocols. The signals provided by anti-CD3 and anti-CD28 mAbs are best delivered to T-cells when the antibodies are immobilized on a solid surface such as plastic plates (Baroja, Lorre et al. 1989; Damle and Doyle 1989) or sepharose beads (Anderson, Blue et al. 1988) (see also U.S. Pat. No. 6,352,694 issued to June, et al.).
A method for immobilizing anti-CD3 and anti-CD28 mAb on tosyl-activated paramagnetic beads with a 4.5 micron diameter and the subsequent use of these beads to stimulate T-cells to proliferate and produce pro-inflammatory cytokines has been described (Levine, Bernstein et al. 1997). It has also been shown that T-cells activated with these beads exhibit properties, such as cytokine production, that make them potentially useful for adoptive immunotherapy (Garlie, LeFever et al. 1999; Shibuya, Wei et al. 2000). These beads are now commercially available from Dynal, NS (Oslo, Norway) under the trade name Dynabeads® CD3/CD28 T-cell Expansion.
The use of paramagnetic beads with immobilized mAbs for expansion of T-cells in cell therapy protocols requires the separation and removal of the beads from the T-cells prior to patient infusion. This is a very labor-intensive process and results in cell loss, cell damage, increased risk of contamination and increased cost of processing. Because of the tight association of the immobilized mAbs on the beads with the corresponding ligands on the surface of the target T-cells, the removal of the beads from the T-cells is difficult. The bead:cell conjugates are often separated by waiting until the T-cells internalize the target antigens and then by using mechanical disruption techniques to separate the beads from the T-cells. This technique can cause damage to the T-cells and can also cause the ligated antigens on the T-cells to be removed from the cell surface for a period of time (Rubbi, Patel et al. 1993). In addition, highly activated T-cells are most desirable for use in cell therapy protocols and T-cells often lose this desirable property during the 24-72 hour waiting time for the T-cells to internalize the target antigens.
The process of removing the paramagnetic beads after separation from the T-cells requires the passing of the cell/bead solution over a magnet. This process can greatly reduce the quantity of beads remaining with the T-cells, but does not completely eliminate the beads. This incomplete bead removal results in some beads being infused in patients which can cause toxic effects. The magnetic bead removal process also reduces the number of T-cells available for therapy, as many T-cells remain associated with the paramagnetic beads even after the waiting time and mechanical disassociation, and are thus removed with the beads in the magnetic field. Some cell loss also occurs when T-cells that may not be bound to the beads become entrapped by beads pulled to the surface next to the magnetic source.